Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-07-07
2003-03-11
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S133000, C558S070000
Reexamination Certificate
active
06531591
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of stable quinone- and photoreactive ketone phosphoramidite reagents designed for automated solid phase synthesis of oligomers terminating in a photoreactive moiety.
BACKGROUND OF THE INVENTION
Attachment of a reporter group or another conjugation to oligonucleotides (ONs) has been the subject of considerable research as the resulting functionalised ONs display great potential as diagnostic or therapeutic agents (S. L. Beaucage,
Comprehensive Natural Products Chemistry
Vol. 7. Ed. E. T. Kool, Editors-in-Chief D. Barton and K. Nakanishi, Pergamon, 1999, 153-250). For example, ONs linked to anthraquinone (anthraquinone-ONs) and derivatives thereof have been prepared with the purpose of increasing the affinity towards complementary ONs via intercalation as well as for studies of site specific modification, cleavage, and crosslinking of duplex structures (K. Mori et al.,
FEBS lett.
1989, 249, 213-218; S. M. Gasper and G. B. Schuster,
J. Am. Chem. Soc.
1997, 119, 12762-12771; L. G. Puskás et al.,
Nucleosides Nucleotides,
1995, 14, 967; H. Kang and S. E. Rokita;
Nucleic acids Res.,
1996, 24, 3896-3902). Another interesting application of anthraquinone-oligomers is the covalent immobilization of oligomers onto polymeric surfaces. Immobilisation of oligomers on various surfaces (Jacobsen, M. H. and Koch, T. WO 96/31557, 1996), such as plastic microtiter plates, microchips and micro particles has been achieved by various means and form the basis for a rapidly expanding technology within the field of diagnostic assays and disease screening assays (F. N. Rehman et al.,
Nucleic acids Res.,
1999, 27, 649-655; P. W. Stevens et al.,
Nucleic acids Res.,
1999, 27, 1719-1727; G. Ramsay,
Nature Biotechnology,
1998, 16, 40-44).
Two general methods for covalent attachment of anthraquinone to oligomers by chemical means have previously been developed. The first method comprises coupling of an activated anthraquinone derivative with a pre-synthesized oligomer containing a reactive group such as a free primary amine function. This approach is illustrated by Kang and Rokita (
Nucleic Acids Res.,
1996, 24, 3896-3902) who synthesized 5′-end anthraquinone-oligodeoxynucleotides (ODNs) for the studies of site-specific and photo-induced alkylation of DNA. A dimethyl-anthraquinone-ODN conjugate was synthesized by coupling of the N-hydroxysuccinimide ester of 2-(3-propionic acid)-1,4-dimethylanthraquinone with 5′-amino hexamethylene linked ODN, obtained by standard automated solid phase synthesis. Anthraquinone-ONs have also been prepared by reaction of ONs containing “amino -linker” modified nucleobases or carbohydrate moieties with activated anthraquinone derivatives (Telser et al.
J. Am. Chem. Soc.
1989, 111, 7226-7232; Akira et al.
Bioconjugate Chem.
1993, 4, 499-508).
The other method comprises converting the anthraquinone into a synthon that can be used for automated solid phase synthesis, e.g. coupling of the anthraquinone to a phosphoramidite reagent. Depending on the availability of the building-block it can be argued that this direct incorporation is the most efficient approach, as the total synthesis of the anthraquinone-oligomers can be performed on an automated synthesizer.
Attachment of anthraquinone derivatives to ONs via direct incorporation has been approached by linking the anthraquinone group to the 2′-O position of a 5′-O-DMT (4,4′-dimethoxytrityl), 3′-O-phosphoramidite nucleoside reagents. K. Yamana et al. (
Bioconjugate Chem.
1996, 7, 715-720) reported the synthesis of 5′-O-dimethoxytrityl 2′-O-(2-anthraquinonylmethyl)uridine 3′-O-cyanoethyl)-N,N-diisopropylphosphoramidite which was used for automated solid phase synthesis of anthraquinone-ONs.
De Mesmaeker et al. (
Bioorganic, Medicinal Chem.
1997, 7, 1869-1874) described the synthesis of nucleoside dimers containing a 3′-5′ amide bond, wherein the nitrogen atom is attached to an anthraquinone molecule through a polymethylene linker. DMT-protection of the 5′-O position and phosphitylation of the 3′-O-position of the dimer afforded a reagent suitable for automated synthesis of anthraquinone-ONs.
A non-basic pseudonucleoside bearing an anthraquinone moiety has been prepared by K.-Y., Lin and M. Matteucci (
Nucleic Acids Res.
1991, 19, 3111-3114, and U.S. Pat. No. 5,214,136). Starting from 2-chloro anthraquinone and diethanol amine an anthraquinone diol derivative was obtained which was converted into a DMT H-phosphonate reagent which was, subsequently, incorporated multiple times into an ODNs.
The above mentioned reagents allow incorporation of an anthraquinone functionality at different positions in an oligomer.
A few examples of phosphoramidite reagents not derived from nudeosides, developed exclusively for incorporation of anthraquinone at the 5′-terminus of an oligomer using automated solid phase synthesis have been reported.
K. Mori et al. (
FEBS Lett.
1989, 249, 213-218) describe the synthesis of anti-HIV active 5′-linked anthraquinone-ODNs wherein an anthraquinone derivative is linked to an oligodeoxynucleotide (ODN) via either an ethylpiperazinyl or a hexamethylene linker. The 5′-linked anthraquinone-ODNs were obtained by coupling of a freshly prepared anthraquinone-ethylpiperazinyl phosphoramidite (obtained in 65% yield) or anthraquinone hexamethylene-linked phosphoramidite to the 5′-end of an ODN sequence using standard automated solid phase synthesis.
The anthraquinone-ethylpiperazinyl phosphoramidite reagent has also been described in WO 90/12802. The anthraquinone phosphoramidite was synthesised using the same procedure as described by K. Mori et al.: 1-chloroanthraquinone was reacted with 1-(2-hydroxyethyl)piperazine affording 1-(1-(2-hydroxyethyl)piperazinyl)anthraquinone which was phosphitylated by N,N-diisopropylphosphoramidochloride in the presence N,N-diisopropylethylamine to afford anthraquinone-ethylpiperazinyl phosphoramidite. The anthraquinone phosphoramidite was used without further purification in the automated solid phase synthesis of 5′-linked anthraquinone-ODNs used for attenuation or destruction of mammalian genetic expression or viral activity.
S. M. Gasperand G. B. Schuster (
J. Am. Chem. Soc.
1997, 119, 12762-12771) described the synthesis of 5′-linked anthraquinone-ODNs with the purpose of establishing the fact that oxidative damage can migrate in double-stranded DNA. For this purpose, two anthraquinone phosphoramidites were synthesised: N-ethyl- and N-pentyl-2-anthraquinonecarboxamide phosphoramidite. The two phosphoramidites were synthesised from anthraquinone-2-carbonyl chloride, which was reacted with 2-amino-1-ethanol or 5-amino-pentanol to afford N-(2-hydroxyethyl)- and N-(5-hydroxypentyl)-2-anthraquinone-carboxamide, respectively. Reaction of these carboxamides with N,N-diisopropylmethyl-phosphonamides chloride afforded the corresponding phosphoramidites as thick dark red oils after column chromatography. Coupling of these anthraquinone phosphoramidites to the 5′-OH terminus of ODNs as the final step in a solid phase synthesis gave anthraquinone-ODN conjugates.
Large scale synthesis of anthraquinone-oligomer conjugates using automated solid phase chemistry requires readily available and relatively stable anthraquinone synthons. Initial attempts to synthesize stable anthraquinone phosphoramidite reagents revealed that the above-mentioned types of reagents appear to be unstable.
The synthesis of an anthraquinone phosphoramidite derivative of N-(6-hydroxyhexyl)-2-anthraquinone carboxamide using N,N,N′,N′-tetraisopropylphosphorodiamidite and tetrazole is described in Example 1. Attempted isolation of this cyanoethyl phosphoramidite led to decomposition. Use of the crude product, after filtration of the reaction mixture, directly onto the DNA synthesizer within one day also led to decomposition. Following, attempts to prepare a cyanoethyl phosphoramidite analog of the N-(2-hydroxyeth
Corless Peter F.
Edwards & Angell LLP
Exiqon A/S
Owens Howard V.
Rees Dianne M.
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